Neuroblastoma remains an important pediatric disease that results in 15% of overall childhood cancer mortality. Several chromosomal aberrations that contribute to the malignant phenotypes are known, but the genetic events essential to initiate neuroblastoma tumorigenesis have not been discovered. We have shown that hereditary predisposition to develop neuroblastoma is genetically linked to the distal short arm of chromosome 16. In addition, we have demonstrated that hemizygous deletions of the same locus are a common somatically acquired event in sporadic neuroblastomas. Thus, we hypothesize that hereditary neuroblastoma is due to mutations in a tumor suppressor gene (HNB1) located at 16p12-13. We also predict that functional inactivation of this gene is responsible for the initiation of many nonfamilial human neuroblastomas, and perhaps other cancers. Accordingly, we propose to first localize HNB1 to within a one megabase region at 16p12-13 and identify candidates for mutation analysis. This will be accomplished using a multifaceted approach including high-density SNP genotyping, array-based comparative genomic hybridization, and gene expression profiling using oligonucleotide arrays. Second, we will identify HNB1 and characterize the spectrum of germline and somatically acquired mutations in familial and sporadic neuroblastomas. We will also survey a large panel of sporadic neuroblastoma primary tumor samples, as well as other human cancers with particular emphasis on neural crest derived tumors, for HNB1 mutations and loss of functional protein expression. Third, we will characterize HNB1 as a tumor suppressor and reintroduce wild-type HNB1 into HNB1-null neuroblastoma cell lines to determine effect on proliferation in vitro and tumorigenicity in vivo. Fourth, we will describe the phenotype of mice harboring heterozygous inactivation of one allele of the murine HNB1 homologue, with particular attention to neural crest tumor formation. Successful completion of these experiments will determine if inactivation of HNB1 is the seminal initiating event for human neuroblastomas, or if genetic heterogeneity exists. Successful completion of this project should dramatically improve our understanding of the fundamental genetic basis of neuroblastoma, and perhaps other human cancers. We also expect that this project will result in a valuable mouse model of this enigmatic pediatric disease. Ultimately, these experiments should lead to the identification of a common pathway to neuroblastoma tumorigenesis that will be an outstanding target for rationally designed therapeutics.